基本情報

所属

• 理学研究院　生物科学部門　行動神経生物学分野

職名

• 教授

学位

• 博士（医学）（2005年01月 東京医科歯科大学）

ホームページURL

http://www.wada-lab.org/

科研費研究者番号

• 70451408

J-Global ID

• 201101094103787000

プロフィール

• 音声発声学習（聞いた声を自分で発声できるようになる）は、ヒトの言語獲得の基盤となる学習です。しかし、非常に限られた動物種のみができる学習様式でもあります。この音声発声学習を可能にする脳神経回路・メカニズムを物質・遺伝子レベルで明らかにしていこうと日々学生たちと格闘しております。研究戦略としては、親鳥の囀りパターンを学習する小鳥（鳴禽類 songbirdソングバード）を動物モデルとして用い、分子生物学・神経生物学・動物行動学をベースに学際的な研究を行っています。また、学習発達過程で、遺伝要因と環境要因の両方の影響を受けながら、いかにして行動レベルの個体差・個性が現れてくるのかということを、ゲノム発現という観点からも研究を進めています。ソングバードを用いた研究ゆえにはっきりと見えてくる重要な生命現象をとらえて離さない、そんな研究を地道だけど、着実に進めていく研究室を運営していきたいと考えています。当研究室での研究に興味がある方は和多までご連絡ください。

研究キーワード

• エピジェネティクス   神経活動依存的遺伝子発現   言語   種特異的行動   発声行動   遺伝子発現   動物行動   学習臨界期   神経可塑性   ソングバード   発声学習   個体差   

研究分野

• ライフサイエンス / ゲノム生物学
• ライフサイエンス / システムゲノム科学
• ライフサイエンス / 神経科学一般
• ライフサイエンス / 動物生理化学、生理学、行動学

担当教育組織

•  学士課程　理学部
•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2021年10月 - 現在 北海道大学 大学院理学研究院 生物科学部門 教授
• 2010年04月 - 2021年09月 北海道大学 大学院理学研究院 生物科学部門 准教授
• 2007年04月 - 2010年03月 北海道大学 大学院先端生命科学研究院 准教授
• 2002年 - 2007年03月 デューク大学 医療センター 神経生物学部門 博士研究員

研究活動情報

論文

• A predisposed motor bias shapes individuality in vocal learning

  Noriyuki Toji, Azusa Sawai, Hongdi Wang, Yu Ji, Rintaro Sugioka, Yasuhiro Go, Kazuhiro Wada

  Proceedings of the National Academy of Sciences 121 3 e2308837121  2024年01月10日 [査読有り][通常論文]
   

  Abstract The emergence of individuality during learned behavior is a general feature of animal species, yet the biological bases of its development remain unknown. Similar to human speech, songbirds develop individually-unique songs with species-specific traits through vocal learning. By taking advantage of songbirds as a model system for studying the neural basis of vocal learning and development, we utilized F₁ hybrid songbirds (Taeniopygia guttata cross with T. bichenovii) to examine the developmental and molecular mechanisms underlying individuality in vocal learning. When tutoring with songs from both parental species, F₁ pupils showed vast individual differences in their acquired songs. Approximately 30% of F₁ hybrids selectively learned either song of the two parental species, whereas others developed merged songs between the parental species. Vocal acoustic biases during vocal babbling were initially observed as individual differences in songs among F₁ juveniles, which were maintained through the sensitive period of song vocal learning. These individual differences in vocal acoustic biases appeared independently from the auditory experience of hearing biological farther’s and passive tutored songs. Furthermore, the idiosyncratic traits of F₁ hybrids’ songs were not correlated with peripheral vocal organ morphology. However, we identified unique transcriptional signatures from the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei associated with individual differences in the acoustic vocal biases, even at the initial stage of vocal learning. These results indicate that a predisposed motor bias influences the individuality observed when learning new motor skills.
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• Dosage compensation of Z sex chromosome genes in avian fibroblast cells

  Ruslan Deviatiiarov, Hiroki Nagai, Galym Ismagulov, Anastasia Stupina, Kazuhiro Wada, Shinji Ide, Noriyuki Toji, Heng Zhang, Woranop Sukparangsi, Sittipon Intarapat, Oleg Gusev, Guojun Sheng

  Genome Biology 24 1 2023年09月20日 [査読有り]
   

  Abstract In birds, sex is genetically determined; however, the molecular mechanism is not well-understood. The avian Z sex chromosome (chrZ) lacks whole chromosome inactivation, in contrast to the mammalian chrX. To investigate chrZ dosage compensation and its role in sex specification, we use a highly quantitative method and analyze transcriptional activities of male and female fibroblast cells from seven bird species. Our data indicate that three fourths of chrZ genes are strictly compensated across Aves, similar to mammalian chrX. We also present a complete list of non-compensated chrZ genes and identify Ribosomal Protein S6 (RPS6) as a conserved sex-dimorphic gene in birds.
• Fetal blockade of nicotinic acetylcholine transmission causes autism-like impairment of biological motion preference in the neonatal chick

  Toshiya Matsushima, Momoko Miura, Nina Patzke, Noriyuki Toji, Kazuhiro Wada, Yukiko Ogura, Koichi J Homma, Paola Sgadò, Giorgio Vallortigara

  Cerebral Cortex Communications 2022年11月18日 [査読有り]
   

  Abstract Several environmental chemicals are suspected risk factors for autism spectrum disorder (ASD), including valproic acid (VPA) and pesticides acting on nicotinic acetylcholine receptors (nAChRs), if administered during pregnancy. However, their target processes in fetal neuro-development are unknown. We report that the injection of VPA into the fetus impaired imprinting to an artificial object in neonatal chicks, while a predisposed preference for biological motion (BM) remained intact. Blockade of nAChRs acted oppositely, sparing imprinting and impairing BM preference. Beside ketamine and tubocurarine, significant effects of imidacloprid (a neonicotinoid insecticide) appeared at a dose ≤1 ppm. In accord with the behavioral dissociations, VPA enhanced histone acetylation in the primary cell culture of fetal telencephalon, whereas ketamine did not. VPA reduced the brain weight and the ratio of NeuN-positive cells (matured neurons) in the telencephalon of hatchlings, whereas ketamine/tubocurarine did not. Despite the distinct underlying mechanisms, both VPA and nAChR blockade similarly impaired imprinting to biological image composed of point-light animations. Furthermore, both impairments were abolished by postnatal bumetanide treatment, suggesting a common pathology underlying the social attachment malformation. Neurotransmission via nAChR is thus critical for the early social bond formation, which is hindered by ambient neonicotinoids through impaired visual predispositions for animate objects.
• Nicotinic acetylcholine receptors in a songbird brain.

  Norman Chinweike Asogwa, Noriyuki Toji, Ziwei He, Chengru Shao, Yukino Shibata, Shoji Tatsumoto, Hiroe Ishikawa, Yasuhiro Go, Kazuhiro Wada

  The Journal of comparative neurology 530 11 1966 - 1991 2022年08月01日 [査読有り][通常論文]
   

  Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission and cell signaling, which contribute to learning, memory, and the execution of motor skills. Birdsong is a complex learned motor skill in songbirds. Although the existence of 15 nAChR subunits has been predicted in the avian genome, their expression patterns and potential contributions to song learning and production have not been comprehensively investigated. Here, we cloned all the 15 nAChR subunits (ChrnA1-10, B2-4, D, and G) from the zebra finch brain and investigated the mRNA expression patterns in the neural pathways responsible for the learning and production of birdsong during a critical period of song learning. Although there were no detectable hybridization signals for ChrnA1, A6, A9, and A10, the other 11 nAChR subunits were uniquely expressed in one or more major subdivisions in the song nuclei of the songbird brain. Of these 11 subunits, ChrnA3-5, A7, and B2 were differentially regulated in the song nuclei compared with the surrounding anatomically related regions. ChrnA5 was upregulated during the critical period of song learning in the lateral magnocellular nucleus of the anterior nidopallium. Furthermore, single-cell RNA sequencing revealed ChrnA7 and B2 to be the major subunits expressed in neurons of the vocal motor nuclei HVC and robust nucleus of the arcopallium, indicating the potential existence of ChrnA7-homomeric and ChrnB2-heteromeric nAChRs in limited cell populations. These results suggest that relatively limited types of nAChR subunits provide functional contributions to song learning and production in songbirds.
• Seasonal regulation of singing-driven gene expression associated with song plasticity in the canary, an open-ended vocal learner

  Shin Hayase, Chengru Shao, Masahiko Kobayashi, Chihiro Mori, Wan-chun Liu, Kazuhiro Wada

  Molecular Brain 14 1 160 - 160 2021年11月 [査読有り]
   

  Songbirds are one of the few animal taxa that possess vocal learning abilities. Different species of songbirds exhibit species-specific learning programs during song acquisition. Songbirds with open-ended vocal learning capacity, such as the canary, modify their songs during adulthood. Nevertheless, the neural molecular mechanisms underlying open-ended vocal learning are not fully understood. We investigated the singing-driven expression of neural activity-dependent genes (Arc, Egr1, c-fos, Nr4a1, Sik1, Dusp6, and Gadd45β) in the canary to examine a potential relationship between the gene expression level and the degree of seasonal vocal plasticity at different ages. The expression of these genes was differently regulated throughout the critical period of vocal learning in the zebra finch, a closed-ended song learner. In the canary, the neural activity-dependent genes were induced by singing in the song nuclei throughout the year. However, in the vocal motor nucleus, the robust nucleus of the arcopallium (RA), all genes were regulated with a higher induction rate by singing in the fall than in the spring. The singing-driven expression of these genes showed a similar induction rate in the fall between the first year juvenile and the second year adult canaries, suggesting a seasonal, not age-dependent, regulation of the neural activity-dependent genes. By measuring seasonal vocal plasticity and singing-driven gene expression, we found that in RA, the induction intensity of the neural activity-dependent genes was correlated with the state of vocal plasticity. These results demonstrate a correlation between vocal plasticity and the singing-driven expression of neural activity-dependent genes in RA through song development, regardless of whether a songbird species possesses an open- or closed-ended vocal learning capacity.
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• Phylogeny and mechanisms of shared hierarchical patterns in birdsong

  Logan S. James, Chihiro Mori, Kazuhiro Wada, Jon T. Sakata

  Current Biology 31 13 2796 - 2808 2021年07月 [査読有り]
  
• Identification of novel avian and mammalian deltaviruses provides new insights into deltavirus evolution

  Masashi Iwamoto, Yukino Shibata, Junna Kawasaki, Shohei Kojima, Yung-Tsung Li, Shingo Iwami, Masamichi Muramatsu, Hui-Lin Wu, Kazuhiro Wada, Keizo Tomonaga, Koichi Watashi, Masayuki Horie

  Virus Evolution 7 1 veab003  2021年02月 [査読有り]
   

  Hepatitis delta virus (HDV) is a satellite virus that requires hepadnavirus envelope proteins for its transmission. Although recent studies identified HDV-related deltaviruses in certain animals, the evolution of deltaviruses, such as the origin of HDV and the mechanism of its coevolution with its helper viruses, is unknown, mainly because of the phylogenetic gaps among deltaviruses. Here, we identified novel deltaviruses of passerine birds, woodchucks, and white-tailed deer by extensive database searches and molecular surveillance. Phylogenetic and molecular epidemiological analyses suggest that HDV originated from mammalian deltaviruses and the past interspecies transmission of mammalian and passerine deltaviruses. Further, metaviromic and experimental analyses suggest that the satellite–helper relationship between HDV and hepadnavirus was established after the divergence of the HDV lineage from non-HDV mammalian deltaviruses. Our findings enhance our understanding of deltavirus evolution, diversity, and transmission, indicating the importance of further surveillance for deltaviruses.
• Manipulations of Sensory Experiences During Development Reveal Mechanisms Underlying Vocal Learning Biases in Zebra Finches

  James LS, Davies R Jr, Mori C, Wada K, Sakata JT

  Developmental Neurobiology 80 132 - 146 2020年03月 [査読有り][通常論文]
  
• Statistical learning for vocal sequence acquisition in a songbird.

  James LS, Sun H, Wada K, Sakata JT.

  Scientific Reports 10 1 2248  2020年02月 [査読有り][通常論文]
  
• Corticobasal ganglia projecting neurons are required for juvenile vocal learning but not for adult vocal plasticity in songbirds

  Miguel Sánchez-Valpuesta, Yumeno Suzuki, Yukino Shibata, Noriyuki Toji, Yu Ji, Nasiba Afrin, Chinweike Norman Asogwa, Ippei Kojima, Daisuke Mizuguchi, Satoshi Kojima, Kazuo Okanoya, Haruo Okado, Kenta Kobayashi, Kazuhiro Wada

  Proceedings of the National Academy of Sciences 116 45 22833 - 22843 2019年11月05日 [査読有り][通常論文]
   

  Birdsong, like human speech, consists of a sequence of temporally precise movements acquired through vocal learning. The learning of such sequential vocalizations depends on the neural function of the motor cortex and basal ganglia. However, it is unknown how the connections between cortical and basal ganglia components contribute to vocal motor skill learning, as mammalian motor cortices serve multiple types of motor action and most experimentally tractable animals do not exhibit vocal learning. Here, we leveraged the zebra finch, a songbird, as an animal model to explore the function of the connectivity between cortex-like (HVC) and basal ganglia (area X), connected by HVC_(X) projection neurons with temporally precise firing during singing. By specifically ablating HVC_(X) neurons, juvenile zebra finches failed to copy tutored syllable acoustics and developed temporally unstable songs with less sequence consistency. In contrast, HVC_(X)-ablated adults did not alter their learned song structure, but generated acoustic fluctuations and responded to auditory feedback disruption by the introduction of song deterioration, as did normal adults. These results indicate that the corticobasal ganglia input is important for learning the acoustic and temporal aspects of song structure, but not for generating vocal fluctuations that contribute to the maintenance of an already learned vocal pattern.
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• Transcriptional regulatory divergence underpinning species-specific learned vocalization in songbirds.

  Wang H, Sawai A, Toji N, Sugioka R, Shibata Y, Suzuki Y, Ji Y, Hayase S, Akama S, Sese J, Wada K

  PLoS Biology 17 11 e3000476  2019年11月 [査読有り][通常論文]
   

  Learning of most motor skills is constrained in a species-specific manner. However, the proximate mechanisms underlying species-specific learned behaviors remain poorly understood. Songbirds acquire species-specific songs through learning, which is hypothesized to depend on species-specific patterns of gene expression in functionally specialized brain regions for vocal learning and production, called song nuclei. Here, we leveraged two closely related songbird species, zebra finch, owl finch, and their interspecific first-generation (F1) hybrids, to relate transcriptional regulatory divergence between species with the production of species-specific songs. We quantified genome-wide gene expression in both species and compared this with allele-specific expression in F1 hybrids to identify genes whose expression in song nuclei is regulated by species divergence in either cis- or trans-regulation. We found that divergence in transcriptional regulation altered the expression of approximately 10% of total transcribed genes and was linked to differential gene expression between the two species. Furthermore, trans-regulatory changes were more prevalent than cis-regulatory and were associated with synaptic formation and transmission in song nucleus RA, the avian analog of the mammalian laryngeal motor cortex. We identified brain-derived neurotrophic factor (BDNF) as an upstream mediator of trans-regulated genes in RA, with a significant correlation between individual variation in BDNF expression level and species-specific song phenotypes in F1 hybrids. This was supported by the fact that the pharmacological overactivation of BDNF receptors altered the expression of its trans-regulated genes in the RA, thus disrupting the learned song structures of adult zebra finch songs at the acoustic and sequence levels. These results demonstrate functional neurogenetic associations between divergence in region-specific transcriptional regulation and species-specific learned behaviors.
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• Auditory-Motor Matching in Vocal Recognition and Imitative Learning.

  Tramacere A, Wada K, Okanoya K, Iriki A, Ferrari PF.

  Neuroscience 409 222 - 234 2019年06月 [査読有り][通常論文]
  
• Recurrent development of song idiosyncrasy without auditory inputs in the canary, an open-ended vocal learner

  Chihiro Mori, Wan-Chun Liu, Kazuhiro Wada

  Scientific Reports 8 1 8732  2018年12月01日 [査読有り][通常論文]
   

  Complex learned behaviors, like bird song and human speech, develop under the influence of both genetic and environmental factors. Accordingly, learned behaviors comprise species specificity and individual variability. Auditory information plays a critical role in vocal learning by songbirds, both to memorize tutor songs and to monitor own vocalizations. Nevertheless, audition-deprived songbirds develop structured, species-specific song patterns. It remains to be elucidated how the auditory input contributes to the development of individual variability of song characteristics. Here we show that an open-ended vocal learner, the canary, annually recapitulates individually unique songs without audition. Although the total number of syllable types was reduced by auditory deprivation, other vocal phenotypes examined in the syllable, phrase, and syntax of songs were conserved between the 1st and 2nd years, both in deafened and intact birds. In deafened canaries, approximately 60% of the syllables were yearly reproduced with consistent acoustic features, whereas the remaining syllables were replaced with new ones in an annual cycle of song development. These results indicate that the open-ended vocal learning of canaries involves an audition-independent mechanism for the development of recurrent song idiosyncrasy.
• Vocal practice regulates singing activity–dependent genes underlying age-independent vocal learning in songbirds

  Hayase S, Wang H, Ohgushi E, Kobayashi M, Mori C, Horita H, Mineta K, Liu WC, Wada K

  PLoS Biology 16 9 e2006537  2018年09月 [査読有り][通常論文]
   

  The development of highly complex vocal skill, like human language and bird songs, is underlain by learning. Vocal learning, even when occurring in adulthood, is thought to largely depend on a sensitive/critical period during postnatal development, and learned vocal patterns emerge gradually as the long-term consequence of vocal practice during this critical period. In this scenario, it is presumed that the effect of vocal practice is thus mainly limited by the intrinsic timing of age-dependent maturation factors that close the critical period and reduce neural plasticity. However, an alternative, as-yet untested hypothesis is that vocal practice itself, independently of age, regulates vocal learning plasticity. Here, we explicitly discriminate between the influences of age and vocal practice using a songbird model system. We prevented zebra finches from singing during the critical period of sensorimotor learning by reversible postural manipulation. This enabled to us to separate lifelong vocal experience from the effects of age. The singing-prevented birds produced juvenile-like immature song and retained sufficient ability to acquire a tutored song even at adulthood when allowed to sing freely. Genome-wide gene expression network analysis revealed that this adult vocal plasticity was accompanied by an intense induction of singing activity-dependent genes, similar to that observed in juvenile birds, rather than of age-dependent genes. The transcriptional changes of activity-dependent genes occurred in the vocal motor robust nucleus of the arcopallium (RA) projection neurons that play a critical role in the production of song phonology. These gene expression changes were accompanied by neuroanatomical changes: dendritic spine pruning in RA projection neurons. These results show that self-motivated practice itself changes the expression dynamics of activity-dependent genes associated with vocal learning plasticity and that this process is not tightly linked to age-dependent maturational factors.
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• Inter- and intra-specific differences in muscarinic acetylcholine receptor expression in the neural pathways for vocal learning in songbirds

  Asogwa NC, Mori C, Sánchez-Valpuesta M, Hayase S, Wada K

  Journal of Comparative Neurology 8 1 8732  2018年09月 [査読有り][通常論文]
  
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• Neurotensin and neurotensin receptor 1 mRNA expression in song-control regions changes during development in male zebra finches

  Devin P. Merullo, Chinweike N. Asogwa, Miguel Sanchez-Valpuesta, Shin Hayase, Bikash R. Pattnaik, Kazuhiro Wada, Lauren V. Riters

  Developmental Neurobiology 78 7 671 - 686 2018年07月01日 [査読有り][通常論文]
   

  Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine-related measures change throughout development in song-control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song-control regions of male zebra finches in four stages of the song learning process: pre-subsong (25 days posthatch dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre-subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671–686, 2018.
• Singing activity-driven Arc expression associated with vocal acoustic plasticity in juvenile songbird

  Hayase S, Wada K

  European Journal of Neuroscience 48 2 1728 - 1742 2018年07月 [査読有り][通常論文]
  
• Sex Differences in Brain Thyroid Hormone Levels during Early Post-Hatching Development in Zebra Finch (Taeniopygia guttata)

  Shinji Yamaguchi, Shin Hayase, Naoya Aoki, Akihiko Takehara, Jun Ishigohoka, Toshiya Matsushima, Kazuhiro Wada, Koichi J. Homma

  PLOS ONE 12 1 e0169643  2017年01月 [査読有り][通常論文]
   

  Thyroid hormones are closely linked to the hatching process in precocial birds. Previously, we showed that thyroid hormones in brain had a strong impact on filial imprinting, an early learning behavior in newly hatched chicks; brain 3,5,3'-triiodothyronine (T-3) peaks around hatching and imprinting training induces additional T3 release, thus, extending the sensitive period for imprinting and enabling subsequent other learning. On the other hand, blood thyroid hormone levels have been reported to increase gradually after hatching in altricial species, but it remains unknown how the brain thyroid hormone levels change during post-hatching development of altricial birds. Here, we determined the changes in serum and brain thyroid hormone levels of a passerine songbird species, the zebra finch using radioimmunoassay. In the serum, we found a gradual increase in thyroid hormone levels during post-hatching development, as well as differences between male and female finches. In the brain, there was clear surge in the hormone levels during development in males and females coinciding with the time of fledging, but the onset of the surge of thyroxine (T-4) in males preceded that of females, whereas the onset of the surge of T-3 in males succeeded that of females. These findings provide a basis for understanding the functions of thyroid hormones during early development and learning in altricial birds.
• A quantitative method for analyzing species-specific vocal sequence pattern and its developmental dynamics

  Raimu Imai, Azusa Sawai, Shin Hayase, Hiroyuki Furukawa, Chinweike Norman Asogwa, Miguel Sanchez, Hongdi Wang, Chihiro Mori, Kazuhiro Wada

  JOURNAL OF NEUROSCIENCE METHODS 271 25 - 33 2016年09月 [査読有り][通常論文]
   

  Background: Songbirds are a preeminent animal model for understanding the neural basis underlying the development and evolution of a complex learned behavior, bird song. However, only a few quantitative methods exist to analyze these species-specific sequential behaviors in multiple species using the same calculation method. New method: We report a method of analysis that focuses on calculating the frequency of characteristic syllable transitions in songs. This method comprises two steps: The first step involves forming correlation matrices of syllable similarity scores, named syllable similarity matrices (SSMs); these are obtained by calculating the round-robin comparison of all the syllables in two songs, while maintaining the sequential order of syllables in the songs. In the second step, each occurrence rate of three patterns of binarized "2 rows x 2 columns" cells in the SSMs is calculated to extract information on the characteristic syllable transitions. Results: The SSM analysis method allowed obtaining species-specific features of song patterns and intraspecies individual variability simultaneously. Furthermore, it enabled quantitative tracking of the developmental trajectory of the syllable sequence patterns. Comparison with existing method: This method enables us to extract the species-specific song patterns and dissect the regulation of song syntax development without human-biased procedures for syllable identification. This method can be adapted to study the acoustic communication systems in several animal species, such as insects and mammals. Conclusions: This present method provides a comprehensive qualitative approach for understanding the regulation of species specificity and its development in vocal learning. (C) 2016 Elsevier B.V. All rights reserved.
• Familial bias and auditory feedback regulation of vocal babbling patterns during early song development

  Daisuke Sato, Chihiro Mori, Azusa Sawai, Kazuhiro Wada

  SCIENTIFIC REPORTS 6 30323  2016年07月 [査読有り][通常論文]
   

  Learned vocalizations are a crucial acoustic biosignal conveying individual traits in many species. Songbirds learn song patterns by listening to a tutor song and performing vocal practice during a sensitive developmental period. However, when and how individual differences in song patterns develop remain unknown. Here, we report that individual differences in vocal output exist even at the earliest song development stage, called subsong. Experiments involving the manipulation of both breeding pairs and song tutoring conditions revealed that the parental pair combination contributes to generating familial differences in syllable duration and variability in the subsong of offspring. Furthermore, after deafening, juveniles immediately changed their subsong by shortening the syllable durations but maintained the individual variability of their subsong temporal patterns, suggesting both auditory-sensitive modification and independent intrinsic regulation of vocal output. These results indicate that the temporal patterns of subsong are not merely disordered vocalization but are regulated by familial bias with sensitivity to auditory feedback, thus generating individual variability at the initiation of vocal development.
• Songbird: a unique animal model for studying the molecular basis of disorders of vocal development and communication

  Chihiro Mori, Kazuhiro Wada

  EXPERIMENTAL ANIMALS 64 3 221 - 230 2015年07月 [査読有り][通常論文]
   

  Like humans, songbirds are one of the few animal groups that learn vocalization. Vocal learning requires coordination of auditory input and vocal output using auditory feedback to guide one's own vocalizations during a specific developmental stage known as the critical period. Songbirds are good animal models for understand the neural basis of vocal learning, a complex form of imitation, because they have many parallels to humans with regard to the features of vocal behavior and neural circuits dedicated to vocal learning. In this review, we will summarize the behavioral, neural, and genetic traits of birdsong. We will also discuss how studies of birdsong can help us understand how the development of neural circuits for vocal learning and production is driven by sensory input (auditory information) and motor output (vocalization).
• Diurnal oscillation of vocal development associated with clustered singing by juvenile songbirds

  Eri Ohgushi, Chihiro Mori, Kazuhiro Wada

  JOURNAL OF EXPERIMENTAL BIOLOGY 218 14 2260 - 2268 2015年07月 [査読有り][通常論文]
   

  Spaced practice affects learning efficiency in humans and other animals. However, it is not well understood how spaced practice contributes to learning during development. Here, we show the behavioral significance of singing frequency in song development in a songbird, the zebra finch. Songbirds learn a complex song pattern by trial-and-error vocalizations as self-motivated practice, which is executed over a thousand times per day during the sensitive period of vocal learning. Notably, juveniles generate songs with a high frequency of singing in clusters with dense singing, whereas adults sing with low frequency in short clusters. This juvenile-specific clustered singing was characterized by clear separations of daily time for intense practice and rest. During the epochs of vocal practice in juveniles, the song structure approached that of song produced at the end of the day. In contrast, during the epochs of vocal rest, the structure of juvenile songs regressed toward that of songs produced at the beginning of the day, indicating a dynamic progression and regression of song development over the course of the day. When the singing frequency was manipulated to decrease it at the juvenile stage, the oscillation rate of song development was dramatically reduced. Although the juvenile-specific clustered singing occurred in non-tutored socially isolated birds or those with auditory deprivation, the diurnal oscillation of vocal development was only observed in non-tutored isolated juveniles. These results show the impact of 'self-motivated' vocal practice on diurnal song developmental plasticity, modulated by the amount of vocal output and auditory feedback.
• Audition-Independent Vocal Crystallization Associated with Intrinsic Developmental Gene Expression Dynamics

  Chihiro Mori, Kazuhiro Wada

  JOURNAL OF NEUROSCIENCE 35 3 878 - 889 2015年01月 [査読有り][通常論文]
   

  Complex learned behavior is influenced throughout development by both genetic and environmental factors. Birdsong, like human speech, is a complex vocal behavior acquired through sensorimotor learning and is based on coordinated auditory input and vocal output to mimic tutor song. Song is primarily learned during a specific developmental stage called the critical period. Although auditory input is crucial for acquiring complex vocal patterns, its exact role in neural circuit maturation for vocal learning and production is not well understood. Using audition-deprived songbirds, we examined whether auditory experience affects developmental gene expression in the major elements of neural circuits that mediate vocal learning and production. Compared with intact zebra finches, early-deafened zebra finches showed excessively delayed vocal development, but their songs eventually crystallized. In contrast to the different rates of song development between the intact and deafened birds, developmental gene expression in the motor circuit is conserved in an agedependent manner from the juvenile stage until the older adult stage, even in the deafened birds, which indicates the auditionindependent robustness of gene expression dynamics during development. Furthermore, even after adult deafening, which degrades crystallized song, the deteriorated songs ultimately restabilized at the same point when the early-deafened birds stabilized their songs. These results indicate a genetic program-associated inevitable termination of vocal plasticity that results in audition-independent vocal crystallization.
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• Global View of the Functional Molecular Organization of the Avian Cerebrum: Mirror Images and Functional Columns

  Erich D. Jarvis, Jing Yu, Miriam V. Rivas, Haruhito Horita, Gesa Feenders, Osceola Whitney, Syrus C. Jarvis, Electra R. Jarvis, Lubica Kubikova, Ana E. P. Puck, Connie Siang-Bakshi, Suzanne Martin, Michael McElroy, Erina Hara, Jason Howard, Andreas Pfenning, Henrik Mouritsen, Chun-Chun Chen, Kazuhiro Wada

  JOURNAL OF COMPARATIVE NEUROLOGY 521 16 3614 - 3665 2013年11月 [査読有り][通常論文]
   

  Based on quantitative cluster analyses of 52 constitutively expressed or behaviorally regulated genes in 23 brain regions, we present a global view of telencephalic organization of birds. The patterns of constitutively expressed genes revealed a partial mirror image organization of three major cell populations that wrap above, around, and below the ventricle and adjacent lamina through the mesopallium. The patterns of behaviorally regulated genes revealed functional columns of activation across boundaries of these cell populations, reminiscent of columns through layers of the mammalian cortex. The avian functionally regulated columns were of two types: those above the ventricle and associated mesopallial lamina, formed by our revised dorsal mesopallium, hyperpallium, and intercalated hyperpallium; and those below the ventricle, formed by our revised ventral mesopallium, nidopallium, and intercalated nidopallium. Based on these findings and known connectivity, we propose that the avian pallium has four major cell populations similar to those in mammalian cortex and some parts of the amygdala: 1) a primary sensory input population (intercalated pallium); 2) a secondary intrapallial population (nidopallium/hyperpallium); 3) a tertiary intrapallial population (mesopallium); and 4) a quaternary output population (the arcopallium). Each population contributes portions to columns that control different sensory or motor systems. We suggest that this organization of cell groups forms by expansion of contiguous developmental cell domains that wrap around the lateral ventricle and its extension through the middle of the mesopallium. We believe that the position of the lateral ventricle and its associated mesopallium lamina has resulted in a conceptual barrier to recognizing related cell groups across its border, thereby confounding our understanding of homologies with mammals. J. Comp. Neurol. 521:3614-3665, 2013. (c) 2013 Wiley Periodicals, Inc.
• Differential androgen receptor expression and DNA methylation state in striatum song nucleus Area X between wild and domesticated songbird strains

  Kazuhiro Wada, Shin Hayase, Raimu Imai, Chihiro Mori, Masahiko Kobayashi, Wan-chun Liu, Miki Takahasi, Kazuo Okanoya

  EUROPEAN JOURNAL OF NEUROSCIENCE 38 4 2600 - 2610 2013年08月 [査読有り][通常論文]
   

  In songbirds, a specialized neural system, the song system, is responsible for acquisition and expression of species-specific vocal patterns. We report evidence for differential gene expression between wild and domesticated strains having different learned vocal phenotypes. A domesticated strain of the wild white-rumped munia, the Bengalese finch, has a distinct song pattern with a more complicated syntax than the wild strain. We identified differential androgen receptor (AR) expression in basal ganglia nucleus Area X GABAergic neurons between the two strains, and within different domesticated populations. Differences in AR expression were correlated with the mean coefficient of variation of the inter-syllable duration in the two strains. Differential AR expression in Area X was observed before the initiation of singing, suggesting that inherited and/or early developmental mechanisms may affect expression within and between strains. However, there were no distinct differences in regions upstream of the AR start codon among all the birds in the study. In contrast, an epigenetic modification, DNA methylation state in regions upstream of AR in Area X, was observed to differ between strains and within domesticated populations. These results provide insight into the molecular basis of behavioral evolution through the regulation of hormone-related genes and demonstrate the potential association between epigenetic modifications and behavioral phenotype regulation.
• Rudimentary substrates for vocal learning in a suboscine

  Wan-chun Liu, Kazuhiro Wada, Erich D. Jarvis, Fernando Nottebohm

  NATURE COMMUNICATIONS 4 2013年07月 [査読有り][通常論文]
   

  Vocal learning has evolved in only a few groups of mammals and birds. The key neuroanatomical and behavioural links bridging vocal learners and non-learners are still unknown. Here we show that a non-vocal-learning suboscine, the eastern phoebe, expresses neural and behavioural substrates that are associated with vocal learning in closely related oscine songbirds. In phoebes, a specialized forebrain region in the intermediate arcopallium seems homologous to the oscine song nucleus RA (robust nucleus of arcopallium) by its neural connections, expression of glutamate receptors and singing-dependent immediate-early gene expression. Lesion of this RA-like region induces subtle but consistent song changes. Moreover, the unlearned phoebe song unexpectedly develops through a protracted ontogeny. These features provide the first evidence of forebrain vocal-motor control in suboscines, which has not been encountered in other avian non-vocal-learners, and offer a potential configuration of brain and behaviour from which vocal learning might have evolved.
• Detecting neural activity-dependent immediate early gene expression in the brain

  Kazuhiro Wada, Chun-Chun Chen, Erich D. Jarvis

  Methods in Neuroethological Research 133 - 149 2013年01月01日 [査読有り][通常論文]
   

  In this chapter, we present an in situ hybridization protocol with radioactive probe that has been successfully and easily used on detecting mRNA expression level and patterns, in multiple tissue types and developmental stages. To detect behaviorally regulated, i.e., motor or sensory, mRNA expression of immediate early genes (IEGs) within cells and tissues in vivo, in situ hybridization is a powerful method for discovering neural activity correlations and novel neural structures. Compared with nonradioactive probe methods such as digoxigenin (DIG) labeling, the radioactive probe hybridization method provides a semi-linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. Furthermore, this method allows us high-throughput mRNA expression analysis for 100–200 sides with 400–1,000 tissue sections simultaneously. This method allows identifying the possible significance and function of interested genes in the nervous system under specific behaviors.
• Specialized Motor-Driven dusp1 Expression in the Song Systems of Multiple Lineages of Vocal Learning Birds

  Haruhito Horita, Masahiko Kobayashi, Wan-chun Liu, Kotaro Oka, Erich D. Jarvis, Kazuhiro Wada

  PLOS ONE 7 8 e42173 - e42173 2012年08月 [査読有り][通常論文]
   

  Mechanisms for the evolution of convergent behavioral traits are largely unknown. Vocal learning is one such trait that evolved multiple times and is necessary in humans for the acquisition of spoken language. Among birds, vocal learning is evolved in songbirds, parrots, and hummingbirds. Each time similar forebrain song nuclei specialized for vocal learning and production have evolved. This finding led to the hypothesis that the behavioral and neuroanatomical convergences for vocal learning could be associated with molecular convergence. We previously found that the neural activity-induced gene dual specificity phosphatase 1 (dusp1) was up-regulated in non-vocal circuits, specifically in sensory-input neurons of the thalamus and telencephalon; however, dusp1 was not up-regulated in higher order sensory neurons or motor circuits. Here we show that song motor nuclei are an exception to this pattern. The song nuclei of species from all known vocal learning avian lineages showed motor-driven up-regulation of dusp1 expression induced by singing. There was no detectable motor-driven dusp1 expression throughout the rest of the forebrain after non-vocal motor performance. This pattern contrasts with expression of the commonly studied activity-induced gene egr1, which shows motor-driven expression in song nuclei induced by singing, but also motor-driven expression in adjacent brain regions after non-vocal motor behaviors. In the vocal non-learning avian species, we found no detectable vocalizing-driven dusp1 expression in the forebrain. These findings suggest that independent evolutions of neural systems for vocal learning were accompanied by selection for specialized motor-driven expression of the dusp1 gene in those circuits. This specialized expression of dusp1 could potentially lead to differential regulation of dusp1-modulated molecular cascades in vocal learning circuits.
• Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

  Chun-Chun Chen, Kazuhiro Wada, Erich D. Jarvis

  JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 62 62 e3764  2012年04月 [査読有り][通常論文]
   

  Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988) (1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.
• ヒト以外の動物にことばはあるか：ヒトの言語とソングバードの囀り

  森千紘, 和多和宏

  Journal of Otolaryngology, Head and Neck Surgery 27 8 1161 - 1168 2011年08月 [査読無し][招待有り]
  
• 聴覚入力による音声パターン学習・維持の神経機構：学習で獲得された発声パターンはいかに維持されるか？

  堀田悠人, 和多和宏

  実験医学 29 4 544 - 550 2011年03月 [査読無し][招待有り]
  
• The dusp1 Immediate Early Gene is Regulated by Natural Stimuli Predominantly in Sensory Input Neurons

  Haruhito Horita, Kazuhiro Wada, Miriam V. Rivas, Erina Hara, Erich D. Jarvis

  JOURNAL OF COMPARATIVE NEUROLOGY 518 14 2873 - 2901 2010年07月 [査読有り][通常論文]
   

  Many immediate early genes (IEGs) have activity-dependent induction in a subset of brain subdivisions or neuron types. However, none have been reported yet with regulation specific to thalamic-recipient sensory neurons of the telencephalon or in the thalamic sensory input neurons themselves. Here, we report the first such gene, dual specificity phosphatase 1 (dusp1). Dusp1 is an inactivator of mitogen-activated protein kinase (MAPK), and MAPK activates expression of egrl, one of the most commonly studied IEGs, as determined in cultured cells. We found that in the brain of naturally behaving songbirds and other avian species, hearing song, seeing visual stimuli, or performing motor behavior caused high dusp1 upregulation, respectively, in auditory, visual, and somatosensory input cell populations of the thalamus and thalamic-recipient sensory neurons of the telencephalic pallium, whereas high egr1 upregulation occurred only in subsequently connected secondary and tertiary sensory neuronal populations of these same pathways. Motor behavior did not induce high levels of dusp1 expression in the motor-associated areas adjacent to song nuclei, where egr1 is upregulated in response to movement. Our analysis of dusp1 expression in mouse brain suggests similar regulation in the sensory input neurons of the thalamus and thalamic-recipient layer IV and VI neurons of the cortex. These findings suggest that dusp1 has specialized regulation to sensory input neurons of the thalamus and telencephalon; they further suggest that this regulation may serve to attenuate stimulus-induced expression of egr1 and other IEGs, leading to unique molecular properties of forebrain sensory input neurons. J. Comp. Neurol. 518:2873-2901, 2010. (C) 2010 Wiley-Liss, Inc.
• Dopamine Receptors in a Songbird Brain

  Lubica Kubikova, Kazuhiro Wada, Erich D. Jarvis

  JOURNAL OF COMPARATIVE NEUROLOGY 518 6 741 - 769 2010年03月 [査読有り][通常論文]
   

  Dopamine is a key neuromodulatory transmitter in the brain. It acts through dopamine receptors to affect changes in neural activity, gene expression, and behavior. In songbirds, dopamine is released into the striatal song nucleus Area X, and the levels depend on social contexts of undirected and directed singing. This differential release is associated with differential, expression of activity-dependent genes, such as egr1 (avian zenk), which in mammalian brain are modulated by dopamine receptors. Here we cloned from zebra finch brain cDNAs of all avian dopamine receptors: the D1 (D1A, D1B, D1D) and D2 (D2, D3, D4) families. Comparative sequence analyses of predicted proteins revealed expected phylogenetic relationships, in which the D1 family exists as single exon and the D2 family exists as spliced exon genes. In both zebra finch and chicken, the D1A, D1B, and D2 receptors were highly expressed in the striatum, the D1D and D3 throughout the pallium and within the mesopallium, respectively, and the D4 mainly in the cerebellum. Furthermore, within the zebra finch, all receptors, except for D4, showed differential expression in song nuclei relative to the surrounding regions and developmentally regulated expression that decreased for most receptors during the sensory acquisition and sensorimotor phases of song learning. Within Area X, half of the cells expressed both D1A and D2 receptors, and a higher proportion of the D1A-only-containing neurons expressed egr1 during undirected but not during directed singing. Our findings are consistent with hypotheses that dopamine receptors may be involved in song development and social context-dependent behaviors. J. Comp. Neurol. 518:741-769, 2010. (C) 2009 Wiley-Liss, Inc.
• ソングバードの囀りを制御する神経回路・遺伝子

  今井礼夢, 森千紘, 和多和宏

  生物の科学 遺伝 64 40 - 47 2010年 [査読無し][招待有り]
  
• Variable Food Begging Calls Are Harbingers of Vocal Learning

  Wan-chun Liu, Kazuhiro Wada, Fernando Nottebohm

  PLOS ONE 4 6 2009年06月 [査読有り][通常論文]
   

  Vocal learning has evolved in only a few groups of mammals and birds. The developmental and evolutionary origins of vocal learning remain unclear. The imitation of a memorized sound is a clear example of vocal learning, but is that when vocal learning starts? Here we use an ontogenetic approach to examine how vocal learning emerges in a songbird, the chipping sparrow. The first vocalizations of songbirds, food begging calls, were thought to be innate, and vocal learning emerges later during subsong, a behavior reminiscent of infant babbling. Here we report that the food begging calls of male sparrows show several characteristics associated with learned song: male begging calls are highly variable between individuals and are altered by deafening; the production of food begging calls induces c-fos expression in a forebrain motor nucleus, RA, that is involved with the production of learned song. Electrolytic lesions of RA significantly reduce the variability of male calls. The male begging calls are subsequently incorporated into subsong, which in turn transitions into recognizable attempts at vocal imitation. Females do not sing and their begging calls are not affected by deafening or RA lesion. Our results suggest that, in chipping sparrows, intact hearing can influence the quality of male begging calls, auditory-sensitive vocal variability during food begging calls is the first step in a modification of vocal output that eventually culminates with vocal imitation.
• Early onset of deafening-induced song deterioration and differential requirements of the pallial-basal ganglia vocal pathway

  Haruhito Horita, Kazuhiro Wada, Erich D. Jarvis

  EUROPEAN JOURNAL OF NEUROSCIENCE 28 12 2519 - 2532 2008年12月 [査読有り][通常論文]
   

  Similar to humans, songbirds rely on auditory feedback to maintain the acoustic and sequence structure of adult learned vocalizations. When songbirds are deafened, the learned features of song, such as syllable structure and sequencing, eventually deteriorate. However, the time-course and initial phases of song deterioration have not been well studied, particularly in the most commonly studied songbird, the zebra finch. Here, we observed previously uncharacterized subtle but significant changes to learned song within a few days following deafening. Syllable structure became detectably noisier and silent intervals between song motifs increased. Although song motif sequences remained stable at 2 weeks, as previously reported, pronounced changes occurred in longer stretches of song bout sequences. These included deletions of syllables between song motifs, changes in the frequency at which specific chunks of song were produced and stuttering for birds that had some repetitions of syllables before deafening. Changes in syllable structure and song bout sequence occurred at different rates, indicating different mechanisms for their deterioration. The changes in syllable structure required an intact lateral part but not the medial part of the pallial-basal ganglia vocal pathway, whereas changes in the song bout sequence did not require lateral or medial portions of the pathway. These findings indicate that deafening-induced song changes in zebra finches can be detected rapidly after deafening, that acoustic and sequence changes can occur independently, and that, within this time period, the pallial-basal ganglia vocal pathway controls the acoustic structure changes but not the song bout sequence changes.
• Molecular Mapping of Movement-Associated Areas in the Avian Brain: A Motor Theory for Vocal Learning Origin

  Gesa Feenders, Miriam Liedvogel, Miriam Rivas, Manuela Zapka, Haruhito Horita, Erina Hara, Kazuhiro Wada, Henrik Mouritsen, Erich D. Jarvis

  PLOS ONE 3 3 2008年03月 [査読有り][通常論文]
   

  Vocal learning is a critical behavioral substrate for spoken human language. It is a rare trait found in three distantly related groups of birds-songbirds, hummingbirds, and parrots. These avian groups have remarkably similar systems of cerebral vocal nuclei for the control of learned vocalizations that are not found in their more closely related vocal non-learning relatives. These findings led to the hypothesis that brain pathways for vocal learning in different groups evolved independently from a common ancestor but under pre-existing constraints. Here, we suggest one constraint, a pre-existing system for movement control. Using behavioral molecular mapping, we discovered that in songbirds, parrots, and hummingbirds, all cerebral vocal learning nuclei are adjacent to discrete brain areas active during limb and body movements. Similar to the relationships between vocal nuclei activation and singing, activation in the adjacent areas correlated with the amount of movement performed and was independent of auditory and visual input. These same movement-associated brain areas were also present in female songbirds that do not learn vocalizations and have atrophied cerebral vocal nuclei, and in ring doves that are vocal non-learners and do not have cerebral vocal nuclei. A compilation of previous neural tracing experiments in songbirds suggests that the movement-associated areas are connected in a network that is in parallel with the adjacent vocal learning system. This study is the first global mapping that we are aware for movement-associated areas of the avian cerebrum and it indicates that brain systems that control vocal learning in distantly related birds are directly adjacent to brain systems involved in movement control. Based upon these findings, we propose a motor theory for the origin of vocal learning, this being that the brain areas specialized for vocal learning in vocal learners evolved as a specialization of a pre-existing motor pathway that controls movement.
• Novel experience induces persistent sleep-dependent plasticity in the cortex but not in the hippocampus.

  Ribeiro S, Shi X, Engelhard M, Zhou Y, Zhang H, Gervasoni D, Lin SC, Wada K, Lemos NA, Nicolelis MA

  Frontiers in Neuroscience 1 43 - 55 1 2007年11月 [査読有り][通常論文]
  
• 小鳥のさえずりと神経科学：言語発声の分子基盤の理解に向けて

  和多 和宏

  脳21 10 4 360 - 365 2007年04月 [査読無し][招待有り]
  
• Lateralized activation of Cluster N in the brains of migratory songbirds

  Miriam Liedvogel, Gesa Feenders, Kazuhiro Wada, Nikolaus F. Troje, Erich D. Jarvis, Henrik Mouritsen

  EUROPEAN JOURNAL OF NEUROSCIENCE 25 4 1166 - 1173 2007年02月 [査読有り][通常論文]
   

  Cluster N is a cluster of forebrain regions found in night-migratory songbirds that shows high activation of activity-dependent gene expression during night-time vision. We have suggested that Cluster N may function as a specialized night-vision area in night-migratory birds and that it may be involved in processing light-mediated magnetic compass information. Here, we investigated these ideas. We found a significant lateralized dominance of Cluster N activation in the right hemisphere of European robins (Erithacus rubecula). Activation predominantly originated from the contralateral (left) eye. Garden warblers (Sylvia borin) tested under different magnetic field conditions and under monochromatic red light did not show significant differences in Cluster N activation. In the fairly sedentary Sardinian warbler (Sylvia melanocephala), which belongs to the same phyolgenetic clade, Cluster N showed prominent activation levels, similar to that observed in garden warblers and European robins. Thus, it seems that Cluster N activation occurs at night in all species within predominantly migratory groups of birds, probably because such birds have the capability of switching between migratory and sedentary life styles. The activation studies suggest that although Cluster N is lateralized, as is the dependence on magnetic compass orientation, either Cluster N is not involved in magnetic processing or the magnetic modulations of the primary visual signal, forming the basis for the currently supported light-dependent magnetic compass mechanism, are relatively small such that activity-dependent gene expression changes are not sensitive enough to pick them up.
• A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes

  Kazuhiro Wada, Jason T. Howard, Patrick McConnell, Osceola Whitney, Thierry Lints, Miriam V. Rivas, Haruhito Horita, Michael A. Patterson, Stephanie A. White, Constance Scharff, Sebastian Haesler, Shengli Zhao, Hironobu Sakaguchi, Masatoshi Hagiwara, Toshiyuki Shiraki, Tomoko Hirozane-Kishikawa, Pate Skene, Yoshihide Hayashizaki, Piero Carninci, Erich D. Jarvis

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 41 15212 - 15217 2006年10月 [査読有り][通常論文]
   

  Songbirds have one of the most accessible neural systems for the study of brain mechanisms of behavior. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene-manipulation tools. To overcome these limitations, we constructed 21 regular, normalized, and subtracted full-length cDNA libraries from brains of zebra finches in 57 developmental and behavioral conditions in an attempt to clone as much of the brain transcriptome as possible. From these libraries, approximate to 14,000 transcripts were isolated, representing an estimated 4,738 genes. With the cDNAs, we created a hierarchically organized transcriptome database and a large-scale songbird brain cDNA microarray. We used the arrays to reveal a set of 33 genes that are regulated in forebrain vocal nuclei by singing behavior. These genes clustered into four anatomical and six temporal expression patterns. Their functions spanned a large range of cellular and molecular categories, from signal transduction, trafficking, and structural, to synaptically released molecules. With the full-length cDNAs and a lentiviral vector system, we were able to overexpress, in vocal nuclei, proteins of representative singing-regulated genes in the absence of singing. This publicly accessible resource http://songbirdtranscriptome.net can now be used to study molecular neuroethological mechanisms of behavior.
• Vocal learning and glutamate receptors in songbirds

  Wada K, Hagiwara M

  Clinical Neuroresearch 24 2 167 - 171 2006年02月 [査読無し][招待有り]
  
• Night-vision brain area in migratory songbirds

  H Mouritsen, G Feenders, M Liedvogel, K Wada, ED Jarvis

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 23 8339 - 8344 2005年06月 [査読有り][通常論文]
   

  Twice each year, millions of night-migratory songbirds migrate thousands of kilometers. To find their way, they must process and integrate spatiotemporal information from a variety of cues including the Earth's magnetic field and the night-time starry sky. By using sensory-driven gene expression, we discovered that night-migratory songbirds possess a tight cluster of brain regions highly active only during night vision. This cluster, here named "cluster N," is located at the dorsal surface of the brain and is adjacent to a known visual pathway. In contrast, neuronal activation of cluster N was not increased in nonmigratory birds during the night, and it disappeared in migrants when both eyes were covered. We suggest that in night-migratory songbirds cluster N is involved in enhanced night vision, and that it could be integrating vision-mediated magnetic and/or star compass information for night-time navigation. Our findings thus represent an anatomical and functional demonstration of a specific night-vision brain area.
• Avian brains and a new understanding of vertebrate brain evolution

  ED Jarvis, O Gunturkun, L Bruce, A Csillag, H Karten, W Kuenzel, L Medina, G Paxinos, DJ Perkel, T Shimizu, G Striedter, JM Wild, GF Ball, J Dugas-Ford, SE Durand, GE Hough, S Husband, L Kubikova, DW Lee, CV Mello, A Powers, C Siang, TV Smulders, K Wada, SA White, K Yamamoto, J Yu, A Reiner, AB Butler

  NATURE REVIEWS NEUROSCIENCE 6 2 151 - 159 2005年02月 [査読有り][通常論文]
   

  We believe that names have a powerful influence on the experiments we do and the way in which we think. For this reason, and in the light of new evidence about the function and evolution of the vertebrate brain, an international consortium of neuroscientists has reconsidered the traditional, 100-year-old terminology that is used to describe the avian cerebrum. Our current understanding of the avian brain in particular the neocortex-like cognitive functions of the avian pallium-requires a new terminology that better reflects these functions and the homologies between avian and mammalian brains.
• Differential expression of glutamate receptors in avian neural pathways for learned vocalization

  K Wada, H Sakaguchi, ED Jarvis, M Hagiwara

  JOURNAL OF COMPARATIVE NEUROLOGY 476 1 44 - 64 2004年08月 [査読有り][通常論文]
   

  Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds-parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and -5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest that neural activity and signal transduction in vocal nuclei of vocal learners will be different relative to the surrounding brain areas. (C) 2004 Wiley-Liss, Inc.
• Revised nomenclature for avian telencephalon and some related brainstem nuclei

  A Reiner, DJ Perkel, LL Bruce, AB Butler, A Csillag, W Kuenzel, L Medina, G Paxinos, T Shimizu, G Striedter, M Wild, GF Ball, S Durand, O Gunturkun, DW Lee, CV Mello, A Powers, SA White, G Hough, L Kubikova, TV Smulders, K Wada, J Dugas-Ford, S Husband, K Yamamoto, J Yu, C Siang, ED Jarvis

  JOURNAL OF COMPARATIVE NEUROLOGY 473 3 377 - 414 2004年05月 [査読有り][通常論文]
   

  The standard nomenclature that has been used for many telencephalic and related brainstem structures in birds is based on flawed assumptions of homology to mammals. In particular, the outdated terminology implies that most of the avian telencephalon is a hypertrophied basal ganglia, when it is now clear that most of the avian telencephalon is neurochemically, hodologically, and functionally comparable to the mammalian neocortex, claustrum, and pallial amygdala (all of which derive from the pallial sector of the developing telencephalon). Recognizing that this promotes misunderstanding of the functional organization of avian brains and their evolutionary relationship to mammalian brains, avian brain specialists began discussions to rectify this problem, culminating in the Avian Brain Nomenclature Forum held at Duke University in July 2002, which approved a new terminology for avian telencephalon and some allied brainstem cell groups. Details of this new terminology are presented here, as is a rationale for each name change and evidence for any homologies implied by the new names. Revisions for the brainstem focused on vocal control, catecholaminergic, cholinergic, and basal ganglia-related nuclei. For example, the Forum recognized that the hypoglossal nucleus had been incorrectly identified as the nucleus intermedius in the Karten and Hodos (1967) pigeon brain atlas, and what was identified as the hypoglossal nucleus in that atlas should instead be called the supraspinal nucleus. The locus ceruleus of this and other avian atlases was noted to consist of a caudal noradrenergic part homologous to the mammalian locus coeruleus and a rostral region corresponding to the mammalian A8 dopaminergic cell group. The midbrain dopaminergic cell group in birds known as the nucleus tegmenti pedunculo-pontinus pars compacta was recognized as homologous to the mammalian substantia nigra pars compacta and was renamed accordingly; a group of gamma-aminobutyric acid (GABA)ergic neurons at the lateral edge of this region was identified as homologous to the mammalian substantia nigra pars reticulata and was also renamed accordingly. A field of cholinergic neurons in the rostral avian hindbrain was named the nucleus pedunculopontinus tegmenti, whereas the anterior nucleus of the ansa lenticularis in the avian diencephalon was renamed the subthalamic nucleus, both for their evident mammalian homologues. For the basal (i.e., subpallial) telencephalon, the actual parts of the basal ganglia were given names reflecting their now evident homologues. For example, the lobus parolfactorius and paleostriatum augmentatum were acknowledged to make up the dorsal subdivision of the striatal part of the basal ganglia and were renamed as the medial and lateral striatum. The paleostriaturn primitivum was recognized as homologous to the mammalian globus pallidus and renamed as such. Additionally, the rostroventral part of what was called the lobus parolfactorius was acknowledged as comparable to the mammalian nucleus accumbens, which, together with the olfactory tubercle, was noted to be part of the ventral striatum in birds. A ventral pallidum, a basal cholinergic cell group, and medial and lateral bed nuclei of the stria terminalis were also recognized. The dorsal (i.e., pallial) telencephalic regions that had been erroneously named to reflect presumed homology to striatal parts of mammalian basal ganglia were renamed as part of the pallium, using prefixes that retain most established abbreviations, to maintain continuity with the outdated nomenclature. We concluded, however, that one-to-one (i.e., discrete) homologies with mammals are still uncertain for most of the telencephalic pallium in birds and thus the new pallial terminology is largely devoid of assumptions of one-to-one homologies with mammals. The sectors of the hyperstriatum composing the Wulst (i.e., the hyperstyiatum accessorium intermedium, and dorsale), the hyperstriatum ventrale, the neostriatum, and the archistriatum have been renamed (respectively) the hyperpallium (hypertrophied pallium), the mesopallium (middle pallium), the nidopallium (nest pallium), and the arcopallium (arched pallium). The posterior part of the archistriatum has been renamed the posterior pallial amygdala, the nucleus taeniae recognized as part of the avian amygdala, and a region inferior to the posterior paleostriaturn primitivum included as a subpallial part of the avian amygdala. The names of some of the laminae and fiber tracts were also changed to reflect current understanding of the location of pallial and subpallial sectors of the avian telencephalon. Notably, the lamina medularis dorsalis has been renamed the pallial-subpallial lamina. We urge all to use this new terminology, because we believe it will promote better communication among neuroscientists. (C) 2004 Wiley-Liss, Inc.
• FoxP2 expression in avian vocal learners and non-learners

  S Haesler, K Wada, A Nshdejan, EE Morrisey, T Lints, ED Jarvis, C Scharff

  JOURNAL OF NEUROSCIENCE 24 13 3164 - 3175 2004年03月 [査読有り][通常論文]
   

  Most vertebrates communicate acoustically, but few, among them humans, dolphins and whales, bats, and three orders of birds, learn this trait. FOXP2 is the first gene linked to human speech and has been the target of positive selection during recent primate evolution. To test whether the expression pattern of FOXP2 is consistent with a role in learned vocal communication, we cloned zebra finch FoxP2 and its close relative FoxP1 and compared mRNA and protein distribution in developing and adult brains of a variety of avian vocal learners and non-learners, and a crocodile. We found that the protein sequence of zebra finch FoxP2 is 98% identical with mouse and human FOXP2. In the avian and crocodilian forebrain, FoxP2 was expressed predominantly in the striatum, a basal ganglia brain region affected in patients with FOXP2 mutations. Strikingly, in zebra finches, the striatal nucleus Area X, necessary for vocal learning, expressed more FoxP2 than the surrounding tissue at post-hatch days 35 and 50, when vocal learning occurs. In adult canaries, FoxP2 expression in Area X differed seasonally; more FoxP2 expression was associated with times when song becomes unstable. In adult chickadees, strawberry finches, song sparrows, and Bengalese finches, Area X expressed FoxP2 to different degrees. Non-telencephalic regions in both vocal learning and non-learning birds, and in crocodiles, were less variable in expression and comparable with regions that express FOXP2 in human and rodent brains. We conclude that differential expression of FoxP2 in avian vocal learners might be associated with vocal plasticity.
• The Avian Brain Nomenclature Forum: Terminology for a New Century in Comparative Neuroanatomy.

  Reiner A, Perkel DJ, Bruce LL, Butler AB, Csillag A, Kuenzel W, Medina L, Paxinos G, Shimizu T, Striedter G, Wild M, Ball GF, Durand S, Gütürkün O, Lee DW, Mello CV, Powers A, White SA, Hough G, Kubikova L, Smulders TV, Wada K, Dugas-Ford J, Husband S, Yamamoto K, Yu J, Siang C, Jarvis ED

  Journal of Comparative Neurology 473 E1 - E6 2004年 [査読有り][通常論文]
  
• Novel SR-rich-related protein clasp specifically interacts with inactivated Clk4 and induces the exon EB inclusion of Clk

  R Katsu, H Onogi, K Wada, Y Kawaguchi, M Hagiwara

  JOURNAL OF BIOLOGICAL CHEMISTRY 277 46 44220 - 44228 2002年11月 [査読有り][通常論文]
   

  We identified a novel serine/arginine (SR)-rich-related protein as a binding partner of Clk4 mutant lacking kinase activity (Clk4 K189R) in the two-hybrid screen and designated it Clasp (Clk4-associating SR-related protein). Northern blot analysis revealed that Clasp mRNA was highly expressed in brain, and in situ hybridization of a mouse brain sagittal section hybridized with antisense probes revealed that both Clasp and Clk4 mRNAs were expressed in the hippocampus, the cerebellum, and the olfactory bulb. Two forms of Clasp were produced by a frameshift following alternative splicing. The staining of An HA-tagged short form of Clasp (ClaspS) showed a nucleoplasmic pattern, while the long form of Clasp (ClaspL) was localized as nuclear dots. In vitro protein interaction assay demonstrated that Clk4 K189R was bound to Clasp while wild Clk4 was not. Overexpression of ClaspL, promoted accumulation of Clk4 K189R in the nuclear dots and the exon EB inclusion from CR-1 and CR-2 pre-mRNA of Clk1. These data suggest that Clasp is a binding partner of Clk4 and may be involved in the regulation of the activity of Clk kinase family.
• Identification of methylated proteins by protein arginine N-methyltransferase 1, PRMT1, with a new expression cloning strategy.

  Wada K, Inoue K, Hagiwara M

  Biochimica et Biophysica Acta 1591 1/3 1 - 10 1-3 2002年08月 [査読有り][通常論文]
  
• Cloning of human PRP4 reveals interaction with Clk1

  T Kojima, T Zama, K Wada, H Onogi, M Hagiwara

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 34 32247 - 32256 2001年08月 [査読有り][通常論文]
   

  Prp4 is a protein kinase of Schizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian serine/arginine-rich protein-specific kinases and Clks, whose substrates are serine/arginine-rich proteins. We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts. Northern blot analysis revealed that hPRP4 mRNA was ubiquitously expressed in multiple tissues. The extended NH2-terminal region of hPRP4 contains an arginine/serine-rich domain and putative nuclear localization signals. hPRP4 phosphorylated and interacted with SF2/ASF, one of the essential splicing factors. Indirect immunofluorescence analysis revealed that endogenous hPRP4 was distributed in a nuclear speckled pattern and colocalized with SF2/ASF in HeLa S3 cells. Furthermore, hPRP4 interacted directly with Clk1 on its COOH terminus, and the arginine/serine-rich domain of hPRP4 was phosphorylated by Clk1 in vitro. Overexpression of Clk1 caused redistribution of hPRP4, from the speckled to the diffuse pattern in nucleoplasm, whereas inactive mutant of Clk1 caused no change of hPRP4 localization. These findings suggest that the NH2-terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1.
• SPK-1, a C-elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development

  H Kuroyanagi, T Kimura, K Wada, N Hisamoto, K Matsumoto, M Hagiwara

  MECHANISMS OF DEVELOPMENT 99 1-2 51 - 64 2000年12月 [査読有り][通常論文]
   

  SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
• Novel RING finger proteins, Air1p and Air2p, interact with Hmt1p and inhibit the arginine methylation of Npl3p

  K Inoue, T Mizuno, K Wada, M Hagiwara

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 42 32793 - 32799 2000年10月 [査読有り][通常論文]
   

  Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in the mRNA processing and export and are post-translationally modified by methylation at arginine residues in their arginine-glycine-rich (RGG) domains. We screened the factors that can interact with the RGG domain of Np13p only in the presence of Hmt1p with the two-hybrid system in Saccharomyces cerevisiae, An isolated clone, YIL079, encodes a novel RING finger protein that was not directly bound to Np13p but associated with the N terminus of Hmt1p. Thus, we designated the gene product Air1p (arginine methyltransferase-interacting RING finger protein). Air1p inhibited the Hmt1p-mediated methylation of Np13p in vitro. Overexpression of Air1p repressed the Hmt1p-dependent growth of cells. Since homology searches indicate that the YDL175 gene product has significant identity (45%) with Air1p, we designated the gene AIR2. Air2p also has a RING finger domain and was bound to Hmt1p. Although single disruption of either gene gave no effect on the cell growth, cells lacking Air1p and Air2p grew at an extremely slow rate with accumulated poly(A)(+) RNA in the nucleus. Thus, Air1p and Air2p may affect mRNA transport by regulating the arginine methylation state of heterogeneous nuclear ribonucleoproteins.
• Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain

  H Sakaguchi, K Wada, M Maekawa, T Watsuji, M Hagiwara

  JOURNAL OF NEUROSCIENCE 19 10 3973 - 3981 1999年05月 [査読有り][通常論文]
   

  We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser(119) in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning.
• Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases

  K Wada, H Sato, H Kinoh, M Kajita, H Yamamoto, M Seiki

  GENE 211 1 57 - 62 1998年04月 [査読有り][通常論文]
   

  Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31, -H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs. (C) 1998 Elsevier Science B.V. All rights reserved.

書籍

• Brain Evolution by Design: From Neural Origin to Cognitive Architecture

  Shigeno S, Murakami Y, Nomura T (担当:分担執筆範囲:Molecular Profiling Reveals Insight into Avian Brain Organization and Functional Columnar Commonalities with Mammals)
  Springer 2017年
• Epigenetics and Neuroendocrinology: Clinical Focus on Psychiatry Volume 1

  Spengler D, Binder E (担当:分担執筆範囲:Differential Regulation of Androgen Receptor and DNA Methylation in Songbirds)
  Springer 2016年
• 研究者が教える動物実験 第３巻

  尾崎まみこ 他 日本比較生理生化学会編 (担当:分担執筆)
  共立出版 2015年
• Methods in Neuroethological Research

  Ogawa H, Oka K (担当:分担執筆範囲:Detecting Neural Activity-Dependent Immediate Early Gene Expression in the Brain)
  Springer 2013年
• 生命誌年刊号vol.69-72「遊ぶ」

  中村 桂子 (担当:分担執筆)
  新曜社 2012年
• 行動遺伝学入門： 動物とヒトの“こころ”の科学

  小出 剛, 山元 大輔 (担当:分担執筆)
  裳華房 2011年

その他活動・業績

• 異種間ハイブリッドソングバードを用いた発声学習個体差の神経分子基盤

  柴田ゆき野, 田路矩之, 郷康広, 辰本将司, 石川裕恵, 和多和宏, 和多和宏 日本神経化学会大会抄録集(Web) 65th 2022年
• 鳴禽類における歌の種分化の神経分子メカニズム

  田路矩之, 柴田ゆき野, 辰本将司, 石川裕恵, 郷康広, 郷康広, 郷康広, 和多和宏, 和多和宏 日本神経化学会大会抄録集(Web) 65th 2022年
• ソングバードの囀りを制御する神経回路・遺伝子 (特集 動物行動の遺伝的基盤を解き明かす--さまざまな動物種における行動遺伝学の歴史と現状ならびに将来への展望)

  今井 礼夢, 森 千紘, 和多 和宏 遺伝 64 (6) 40 -47 2010年11月 [査読無し][通常論文]
  
• 研究助成 エピジェネティクス制御による発声学習臨界期中の遺伝子発現機構の検討 (2007年度(第16回)テーマ: エピジェネティクス)

  和多和宏 中山科学振興財団活動報告書 2007 42 -47 2007年 [査読無し][通常論文]
  
• 海外便り・研究室紹介 How long can we study?

  和多和宏 比較生理生化学 23 (3) 170 -172 2006年 [査読無し][通常論文]
  
• 第23回神経科学大会第10回日本神経回路学会合同大会シンポジューム「学習する本能」レポート

  和多 和宏 比較生理生化学 18 (1) 50 -51 2001年04月30日

共同研究・競争的資金等の研究課題

• ソングバードを用いた「吃音」発症メカニズム解明に向けた神経回路機能学的研究

  日本学術振興会：科学研究費助成事業 挑戦的研究(開拓)

  研究期間 : 2021年07月 -2025年03月 

  代表者 : 和多 和宏

   

  本研究目的として、発声学習時の大脳基底核ループ経路の機能異常にあるとする『大脳基底核ループ機能異常』仮説を検証することを目的にしている。これを踏まえ、以下の2点に関して研究を進めた。 （１）本研究でフォーカスする大脳基底核歌神経核（Area X）を構成する全細胞タイプの同定及び、その構成比を検証した。この際、Area Xの主要構成細胞であるmedium spiny neuron（MSN）のサブタイプを明らかにするために、特にドーパミン、ノルアドレナリン、アセチルコリン受容体のサブユニット別分類を実施した。これにより、今後の機能阻害操作を行う対象選別の基礎情報として用いる。また、各細胞タイプ・MSNサブタイプに特異的発現を示す遺伝子群を同定できたため、ATAC-seq実験によりこれらの遺伝子群の発現調節領域を明らかにし、それをArea X内の各細胞タイプ特異的遺伝子操作実験に用いる。 （２）本研究ではアデノ随伴ウイルス（Adeno-associated virus: AAV）による遺伝子改変操作による基底核ループ機能阻害を計画している。当該年度では、CRISPR-saCas9システムによる遺伝子ノックダウンの可能性を検討した。しかし、当研究室で樹立したzebra finch由来繊維芽細胞でのゲノム改変効率に対して、AAV感染による脳内神経細胞における効率は想定していたよりも低く（< 1%）、遺伝子ノックアウト実験として個体レベルの行動解析を実施するのには不向きである結果を得た。これに代わる方法として、通常発現プロモーターによって駆動できるmiR30バックボーンにしたshRNAi法を導入した。その結果、脳内においても、これまでにテストした対象遺伝子群すべてにおいて、発現量の60~85%を低下させる非常に安定した遺伝子ノックダウン法を確立できた。
• 種特異的な発声学習行動生成に関わるゲノム機能の解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2021年04月 -2025年03月 

  代表者 : 和多 和宏

   

  本研究は、種間のゲノム配列上の違いが、いかにして脳内に共通して存在する神経回路における遺伝子発現に影響を与え、その結果、個体レベルにおける種特異的発声学習行動の生成につながり得るのかを明らかにすることを目的としている。当該年度においては、コロナウイルス感染拡大による研究活動制限を受けたが、概ね当初予定した通りの実験を実施できた。また、そこから得有られた研究成果は期待以上のものがあった。その大きな起点になったのが、新規シングルセルRNA-seq解析方法による、複数個体混合サンプルをシークエンス後に個体識別できることに成功したことである。これによって、研究申請時に想定したサンプル数よりも多くシングルセルRNA-seqを施行でき、当初よりも多くの実験サンプル比較解析ができた。当初の研究計画に沿って、鳴禽類ソングバード３種（zebra finch, owl finch, cherry finch）の発声運動回路を構成する歌神経核HVC, RAの新鮮凍結サンプルを用いてシングルセルRNA-seqを実施した。その結果、種特異的遺伝子発現の違いが、今回フォーカスしたすべての種で、グルタミン酸作動性興奮性投射ニューロンに蓄積していること、またその種特異的な遺伝子発現を受けている遺伝子群のなかには、系統進化過程で複数回発現変化を受けている遺伝子が存在することが明らかになった。現在、これらの遺伝子がどの染色体に位置するのか解析を進めている。また、この細胞タイプレベルで観察された種差が歌発声学習臨界期初期には観察されるが、学習臨界期後の成鳥時に観察される発現パターンとは異なるために、発声学習臨界期間中においても遺伝子発現パターンの種特異的なダイナミクスを維持していることを示唆する結果を得た。これは、歌学習発達過程にも種特異的な発声パターンの制御メカニズムが存在することを意味している可能性を示す。
• 言語の下位機能の生物学的実現

  日本学術振興会：科学研究費助成事業

  研究期間 : 2017年06月 -2022年03月 

  代表者 : 岡ノ谷 一夫, 関 義正, 幕内 充, 香田 啓貴, 和多 和宏

   

  鳥を対象とした研究では、セキセイインコにおいて意図共有の萌芽的行動が見られるかを検討するために、個体間での学習性の運動の同調・発声パターンの同調および鳴き交わしのタイミングを分析した。その結果、協調運動を要する課題において一部の個体が能動的に運動パターンを変え得ることが示唆された。また、視覚刺激に対する音によるラベル付け能力を検討したところ、既知個体の姿と声についてであれば、一部のトリはその組み合わせの正誤を弁別できることが示唆された。ジュウシマツの社会的意図を視線から計測する試みが成功し、鳥は注意している対象を正面から注視はせず、60度の向きから注視することがわかった。これをもとに、鳥の頭方向を弁別する訓練を行ったところ、90度を正刺激として強化、0度を負刺激として消去するほうが、その反対より学習が速かった。さえずりの種差が歌運動神経核で発現する遺伝子の種差に対応していることがわかった。 霊長類を対象とした研究では、霊長類の発声が関連する行動や、雌雄間のコミュニケーション、その適応的な意義について、新たに深層学習を中心とした機械学習による分析手法を導入し調べた。動物の歌のみならず、個体間関係の分析を、自然言語処理に用いられる手法を利用して、社会構造に出現する階層的な構造を調べることも可能となった。 ヒトを対象とした研究では、文理解と算術計算にそれぞれ要請される階層構造構築がブローカ野を賦活させることを明らかにした。さらに描画行為にも階層構造構築が関与することを行動実験で示し、その脳基盤がやはりブローカ野であることを示すための実験の準備を進めた。また意図共有の脳メカニズム解明のため意味論的処理を行う際の脳活動をfMRIで計測し、両側頭頂側頭接合領域の相補的活動を発見した。
  
• 発声学習バイアスの個体差形成に関わる脳内遺伝子発現メカニズムの解明

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2019年04月 -2021年03月 

  代表者 : 和多 和宏

   

  本研究目的は、生育環境を統制しても表出してくる『学習行動の個体差』が、脳部位特異的にいかに表象されているのか、単一細胞レベルの遺伝子発現を父・母由来別のゲノム情報を用い、その父母アレルからの読み出しが子の発声行動表現型の個体差形成にいかに関わってくるのかを検証実験により明らかにすることである。そのために当該年度において、歌神経核内単一細胞レベルでのAllelic imbalance制御と学習バイアスの個体差形成との機能相関を明らかにすべく、統制された歌学習環境下においても明確な学習バイアスを示したハイブリッド個体の脳サンプルを用い、歌運動神経核HVC, RAを構成する単一細胞レベルでのsingle nuclei RNA-seq (snRNA-seq)を実施した。既に同定済の種特異的SNPsを用いることで父母アレル発現量比(Allelic imbalance)を算定することにも成功した。現在、各歌神経核内の細胞クラスター（各種投射ニューロン、介在ニューロン等）ごとの父母アレル発現量比と各個体の発声学習バイアスとの機能相関解析を行なっており、細胞タイプによって父・母アレルからの発現比が異なることが起こっていることを明らかにしつつある。この現象が各個体間で異なるのか、それも同様に維持されているのか、今後検証を進める予定である。以上により、歌神経核内の「どの細胞タイプ」で、「どのような遺伝子群」の発現制御が発声学習バイアス度・歌表現型を最もよく説明できるのかを明らかにする。
• 音声発声学習におけるミラーニューロンの機能発現の実験的検証

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 和多 和宏

   

  本研究目的は、十分に理解が進んでいないミラーニューロンの学習・認知行動における神経機能を個体レベルで明らかにすることである。そのために、霊長類以外でミラーニューロンが見つかっている鳴禽類ソングバードの音声-聴覚ミラーニューロン大脳皮質-基底核投射HVC(X)神経細胞に着目した。当該年度の研究では，この大脳皮質-基底核投射神経細胞の機能を明らかにすべく、細胞死（アポトーシス）を人工的に誘導することができるタンパク質をこの神経細胞だけに出して選択的にこの細胞タイルを除去した。このために、ソングバード脳においても逆行性感染能力をもつアデノ随伴ウイルスを開発した。その結果、キンカチョウの発声学習前の若鳥のときにこのHVC(X)神経細胞をなくすと、その後の歌学習がうまくできず、歌の音響特性、及び音素時系列配列の制御に異常が起こり、成鳥になってもキンカチョウ本来の歌パターンでさえずりができないようになった。これに対して、歌学習後の成鳥時から大脳皮質-基底核投射神経細胞をなくすと、学習した歌パターンに変化もなく、またその後聴覚剥奪後の歌の変化も正常個体と同じように起こることが分かった。これらの結果は、大脳皮質-基底核投射HVC(X)神経細胞は、発声学習状態によってその神経回路機能への貢献度が異なっており、発声学習時に時間情報を基底核に送っている可能性が考えられる。今回比較的歌構造が定型的なキンカチョウで得られた結果が、より複雑な歌構造をもつ他の鳥種で同様のことが起こっているのか明らかにすべく現在研究を進めている。
• 鳥類の歌制御の報酬機構と外発的・内発的動機づけ

  日本学術振興会：科学研究費助成事業

  研究期間 : 2017年04月 -2020年03月 

  代表者 : 岡ノ谷 一夫, 池渕 万季, 関 義正, 和多 和宏

   

  鳥のオスはメスに対して求愛のために歌をうたうが、１羽でも歌をうたう。後者は内発的動機づけによって維持されていると考えられるが、その実体は不明である。本研究では、鳴禽類の脳内で動機づけがどのように生成され自発的な発声に至るのかを明らかにするために、中脳ドパミン神経系と大脳基底核の神経活動を計測した。その結果、大脳基底核において、発声を開始する数秒前から緩やかな発火頻度の上昇を示す一群の細胞を見出した。これに対し、腹側被蓋野・黒質において発声の開始時に一過的な神経活動の上昇を示す細胞を見出した。これらより、中脳ドパミン神経が発声開始の最終的な引き金を引くことで発声が始まると考えられる。
• ソングバードを用いた発話コミュニケーション障害「吃音」の神経行動学的研究

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  研究期間 : 2017年06月 -2020年03月 

  代表者 : 和多 和宏

   

  「吃音」の動物モデルとしてソングバードを用い、音声繰り返し異常の神経メカニズムの解明を目標に研究を行った。DlxプロモータによるGABAergic interneuronに限定した遺伝子発現制御を可能し、介在ニューロン特異的なアポトーシスを誘導した。その結果、成鳥においても、繰り返し音素の異常増加が誘導された。これに対して、pan-cell typeプロモータのCMVプロモーターを用いた場合、音素繰り返しの軽微な増加が観察されたのみであった。これは大脳基底核内の細胞タイプ特異的な機能異常による運動パターンの時系列制御の困難さが誘導されるとする『大脳基底核ループ機能異常』仮説を強くサポートする。
• 自発的行動に起因する発声学習表現型の個性創発の神経分子基盤の解明

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2017年04月 -2019年03月 

  代表者 : 和多 和宏

   

  自発的な発声学習行動によって、発達過程における脳内遺伝子発現及び、エピジェネティクス動態がいかに影響を受け、その後の個体レベルでの行動様式の「個性」創発に関わるのか検証することを目的として研究を進めてきた。研究対象としたキンカチョウは、学習臨界期に1日につき数百回以上の発声練習を自発的に繰り返すことにより歌を完成させ、獲得した歌はその後一生涯維持される。しかし、この自発的な発声練習量に個体差が存在する。前回の公募研究では、学習臨界期中の発声練習回数・頻度をコントロールし、人為的に発声行動量の個体差を誘導し、その学習効率及び、学習臨界期への影響を検証した。その結果、学習臨界期中の発声練習量を制御した場合、本来であれば歌パターンが固定化する成鳥になっても、幼鳥のような未熟な歌を出し、さらにその時点からでも発声学習ができることが明らかになった。また、ソングシステムにおける遺伝子発現動態をゲノムワイドに検証した結果、多くの遺伝子群が正常個体と同様に日齢により発現調節されるなか、脳部位（歌神経核RA）特異的に発声練習時にだけに発現誘導され、かつ発声学習の終了と共に読み出されなくなっていく119個の神経活動依存的遺伝子群の存在を明らかにした。これらの結果は、発声学習臨界期制御は、日齢ではなく発声経験の蓄積が重要な行動因子になること、そしてこの自発的に生成される発声行動に個体差が生じる余地があることを意味する。
• 脳内エピジェネティクス変化による運動パターン学習と維持メカニズムの解明

  文部科学省: 科学研究費補助金：新学術領域研究（記憶ダイナミクス）

  研究期間 : 2016年04月 -2018年03月 

  代表者 : 和多 和宏
• ミラーニューロンの形成過程：トリの聴覚発声系をモデルとした学習・発達研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 2014年04月 -2017年03月 

  代表者 : 岡ノ谷 一夫, 和多 和宏, 池渕 万季

   

  模倣学習では、他者の動きを観察し、自己の動きを制御する必要がある。ミラーニューロン(MN)はこの過程で必要とされる神経細胞で、観察と実行の双方で活動する。鳥の高次発声中枢にMNがあるとされているが、それがどう形成されるのかわかっていない。私たちはジュウシマツの高次発声中枢の神経細胞の一部が大脳基底核に投射すること、そしてその細胞の１／４程度が特定の歌要素を聴く際とその歌要素を発する際、いずれでも活動することを発見した。さらに、発達の過程で遺伝子発現の調整に関わる因子を阻害すると歌の学習に異常が生ずることを見いだした。以上の結果は、MNの形成過程でエピジェネティクスが作用することを示唆する。
• Allelic imbalanceによる行動表現型のゲノム拘束性と可塑性の検証

  日本学術振興会：科学研究費助成事業

  研究期間 : 2013年04月 -2016年03月 

  代表者 : 和多 和宏, 峯田 克彦

   

  本研究は、『遺伝要因』を父・母由来別のゲノム情報として分別し、生育過程における『環境要因』を介して学習行動において脳部位特異的にいかに表象されているかを遺伝子発現レベルで明らかにすることを目指し研究を進めてきた。この目的のために、種特異的な囀りパターンを学習によって獲得する鳴禽類ソングバードの異種間ハイブリッド個体を用い、音声発声学習過程における発声行動表現型と脳内遺伝子発現に着目した研究を進めた。その結果、種特異的SNPsを同定し、脳内で種特異的な発現制御を受ける遺伝子群を明らかにした。
• 発声行動依存的な脳内エピジェネティクス動態と学習臨界期制御機構の解明

  文部科学省: 科学研究費補助金：基盤研究(B)

  研究期間 : 2013年04月 -2016年03月 

  代表者 : 和多 和宏
• Allelic Invaraianceによる行動表現型のゲノム拘束性と可塑性の検証：ソングバード異種間ハイブリッドを用いた神経行動学的研究

  文部科学省：科学研究費補助金 挑戦的萌芽研究

  研究期間 : 2013年04月 -2016年03月 

  代表者 : 和多 和宏
• ソングバードを用いた時空間制御を与える遺伝子発現系の開発と行動実験への応用

  日本学術振興会：科学研究費助成事業

  研究期間 : 2011年04月 -2013年03月 

  代表者 : 和多 和宏

   

  本研究は、時期特異的・細胞タイプ特異的なウイルス発現系の確立を通して、鳴禽類ソングバードを音声発声学習研究の動物モデルとして利用していくことを目的とした。具体的にはウイルス発現系を用いたソングバード脳内遺伝子改変技術の開発・実験応用を次の2点にフォーカスして施行した。 (I)「発現時期特異性」と「投射神経細胞特異性」を兼ね備えたウイルス発現実験系の確立と利用：これまでに様々なプロモーター及びウイルス種の組み合わせによる脳内試験投与を行ってきた。その結果、AAV-9(アデノ随伴ウイルス９型）によってRA神経核内投射神経細胞特異性に遺伝子発現誘導が可能となることを見出した。この発現は最低でも２か月以上におよび、ソングバード発声行動学習臨界期間を十分カバーするものであった。現在、エピジェネティクス制御因子関連遺伝子の発現誘導を試みている。 (II) レンチウイルスによるトランスジェニック・ソングバード作製 レンチウイルスを胚発生時の生殖系列細胞に感染させることによりF1世代でトランスジェニック・ソングバード作出をロックフェラー大学Wan-chun Liu博士と共同研究を現在進めている。これまでに、キンカチョウのみならず、カナリアでもトランスジェニック個体を作出することに成功した。カナリアは毎年発声学習の再学習を行う種であり、このトランスジェニック個体作出の成功により発声学習研究の新たな動物モデルとしての可能性をもっている。
• ソングバードを用いた発話コミュニケーション障害「吃音」動物モデルの確立と応用

  文部科学省：科学研究費補助金 挑戦的萌芽研究

  研究期間 : 2010年04月 -2013年03月 

  代表者 : 和多和宏
• ソングバードを用いた時空間制御を与える遺伝子発現系の開発と行動実験への応用

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  研究期間 : 2009年04月 -2013年03月 

  代表者 : 和多和宏

   

  本研究の目的は、時期特異的・細胞種特異的なウイルス発現系の確立を通して、鳴禽類ソングバードを音声発声学習研究の動物モデルとして利用していくことにある。ソングバードの発声学習・生成行動は、特異的な神経回路Song system(ソングシステム)がすでに同定されている点において脊椎動物の中でユニークな神経回路といえる。さらにこのSong systemは、発声運動制御系と発声学習制御系の2つの神経回路に分別することができ、動物の行動発現(発声学習・生成行動)を「運動と学習」の2つの回路機能に分解して検証することが可能である。 これまでに音声発声学習では「声を出す」という自発的行動が、脳内分子レベルにおいても重要な意味をもつと考え、ソングバードの音声発声学習過程において[発声行動依存性]+[神経回路特異性]+[学習臨界期間限定性]を兼ね備えた多段階発現制御を受ける遺伝子群が存在することを明らかにしてきた。これらを踏まえ、当該年度においては、高タイターをもつRV-G(+)lentivirusの作成を続けると同時に、これまでに発声行動によって脳内で発現誘導される3つの遺伝子Arc,Gadd45b,H3.3Bを強制発現、及びRNAiによるdown regulationを可能とするlentivirus plasmidの構築を終え、現在ウイルスを作成中である。 また、細胞種特異的発現を可能とするプロモーター発現調節部位の同定及びそのレンチウイルス発現系への応用を目指し、神経活動依存的でかつ、ソングシステムに限定的に発現誘導される遺伝子(Dusp1)が存在することを明らかにした。その過程で得られた結果の一部を論文(Horita et.Al., J.Comp.Neurol.518,2873-2901(2010))として報告した。現在、さらにminimum regulatory regionを同定する実験を準備でしている。
• 行動の進化を探る:種特異的発声行動の分子基盤の抽出と検証

  文部科学省：科学研究費補助金(若手研究(A))

  研究期間 : 2009年04月 -2012年03月 

  代表者 : 和多和宏

   

  本研究では、「行動の進化」を中心課題に据え、種特異的行動発現に関わる分子基盤の抽出とその神経分子機能としての行動表現型への影響を実験的に検証することを目的とした。そのために、鳴禽類ソングバードの囀り行動とその発声パターンを生成する脳内神経回路に焦点を据えた研究を行い、(1)アジア圏に生息するソングバード10種の分子系統樹作成及び、その発声パターンの種特異的表現型抽出、(2)新規発声パターン解析法の確立、(3)ソングバードDNAアレイによるソングシステムにおける種特異的脳内発現を示す遺伝子群の抽出、(4)ウイルス発現系による外来遺伝子の発現方法の確立を施行した。
• 発声学習臨界期制御に関わる遺伝子群の脳内機能

  文部科学省：科学研究費補助金(特定領域研究)

  研究期間 : 2008年04月 -2010年03月 

  代表者 : 和多和宏

   

  本研究の目的は音声発声学習の臨界期制御に関わる遺伝子群を明らかにし、その脳内機能を実験的に検証していく手立てを確立することにある。音声発声学習では「声を出す」という自発的行動が、脳内分子レベルにおいても重要な意味をもつと考え、これまでに鳴禽類ソングバードの発声行動により発現誘導される40にも上る遺伝子群の網羅的な同定してきた。昨年度においては、さらに[発声行動依存性]+[神経回路特異性]+[学習臨界期間限定性]を兼ね備えた遺伝子群が存在することを明らかにし、ソングバード脳内神経核で、各々の脳部位特異的に多段階発現(時空間)制御を受けた発現制御を同定してきた。これを受け今年度は、[発声行動依存性]+[神経回路特異性]+[学習臨界期間限定性]を兼ね備えた遺伝子群の脳内機能を見るべくレンチウイルスを用いた脳内遺伝子発現系の実験を開始している。また同時に、特に神経細胞特異的に発現誘導することを目指した改良を試みている。またさらに、音声発声学習途上における音素配列の時系列構造を獲得、生成していく過程を数値化していく解析法も開発している。 以上のように、脳内遺伝子群の発達段階・環境に即した多段階発現(時空間)制御を受けた発現制御に着目し、鳴禽類ソングバードを動物モデルとして用い、感覚運動学習の臨界(適応)期制御に関わる神経分子基盤を明らかにすべく研究を進めている。
• 発声学習臨界期制御の脳内分子機序

  文部科学省：科学研究費補助金(特別研究促進費, 基盤研究(C))

  研究期間 : 2007年04月 -2009年03月 

  代表者 : 和多和宏

   

  当該年度において、研究目標の第一に挙げた「発声行動依存性」+「神経回路特異性」+「学習臨界期間限定性」を兼ね備えた発声学習臨界期関連遺伝子群の同定を行ってきた。その結果、鳴禽類キンカチョウの発声パターンの学習中の幼鳥と発声学習を終えた成鳥との間で、発声行動時に異なる発現誘導効率をもつ遺伝子群の同定に成功した。今回、同定された遺伝子の一つにArc/Arg3.1があるが、グルタミン酸受容体のアンカープロテインとしての機能をもつことが知られている。キンカチョウでは、舌下神経核に投射している神経細胞特異的にその発現誘導効率が学習臨界期中のみに高いことを確認できた。これらのことは、同じ発声行動を生成していても、学習臨界期中とその後では、脳内の部位特異性のみならず、細胞種特異的に、神経興奮依存的な遺伝子発現誘導が制御されていることを意味する。現在、DNAメチル化修飾等によるエピジェネティック制御機構との関わりを検証する実験を施行している。 今回同定できた遺伝子群の多くに哺乳類ホモログが存在することを確認できた。上述のArcのノックアウトマウスでも、海馬での長期増強や学習・記憶形成に障害が示されている。これらのことからも、本研究で同定された遺伝子群の動物個体レベルにおける機能を検証することは重要であると考える。音声発声学習・生成の研究は従来使用されてきたマウス、ラットの動物種では不適である...

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